Method for producing triostin

ABSTRACT

A METHOD FOR PRODUCING AN ANTIBIOTIC, TRIOSTIN, HAVING ANTIBACTERIAL PROPERTIES, CHARACTERIZED BY CULTIVATING STREPTOMYCES TRIOSTINICUS, N. SP. ATCC NO. 21,043, IN AN AQUEOUS NUTRIENT MEDIUM UNDER A SUBMERGED AEROBIC CONDITION, RECOVERING FROM THE CULTURED BROTH A TRIOSTIN COMPLEX CONSISTING SUBSTANTIALLY OF TRIOSTIN A, TRIOSTIN B AND TRIOSTIN C AND, IF DESIRED, ISOLATING EACH COMPONENT OF THE TRIOSTIN COMPLEX.

United States Patent fi 3,641,631

Patented Mar. 7, 1972 3,647,631 in the production of the antibiotics, triostins. These and METHOD FOR PRODUCING TRIOSTIN other ob ects of the present invention will become appar- K Katagil-i, 1 i, J assignmto ent to those conversant with the art to which the present shiono 'i & (30., Ltd,, O ak Japan invention is concerned from the following descriptions. No Drawing. Filed Feb. 19, 1968, Ser. No. 706,678 5 The microorganism employed in the method of the Int. Cl. C12d 9/00 present invention was isolated from a soil sample collected 195-80 3 Clalms in Japan, the typical strain of which was deposited with the American Type Culture Collection under an accession number ATCC No. 21,043. This organism shows the fol- ABSTRACT OF THE DISCLOSURE 10 lowing micro-biological characteristics. A method for producing an antibiotic, triostin, having lowmg microblologlcal charactensncs' antibacterial properties, characterized by cultivatin Stre romy'ces triostinicus, n. sp. ATCC No. 21,043: in 5n MORPHOLOGICAL CHARACTERISTICS aqueous nutrient medium under a submerged aerobic con- The morphology of the culture was studied on glucosedition, recovering from the cultured broth a triostin comasparagin agar. The colony is covered with velvety and plex consisting substantially of triostin A, triostin B and gray to grayish white aerial mycelium. The microscopic triostin C and, if desired, isolating each component of observation shows that the aerial mycelium is simply the triostin complex. branched, waved or slightly curved, and occasionally forms spirals or loops. Spores are cylindrical with smooth surface, 0.7 to 1.0 by 5 microns.

CULTURAL CHARACTERISTICS [For 14 days at 27 C.]

Soluble Medium Growth Vegetative mycelium Aerial mycelium pigment Note Czapek agar Poor White to gray None None.... Glucose, Czapek agar. Good Brownish gray do do Asparagin, glucose agar .do White to grayish white Abundant, grayish white. ...d0 Ca-malate agar. Brownish gray Poor, w 'te Starch agar Grayish white..- is Hydrolysis of starch weakly positive. Bouillon agar. Brownish ra None Chromogenicity negative. Glucose bouillon agar inkled. Pale yellowish brown do 0- Peptone, glucose agar. Yellowish brown Poor, white. Tyrosine agar Poor Colorless .do do 'Iyrosinase negative. Egg-yolk .r Yellow None goitlato plug 11 Plaque color colorless.

fie fiffflu do I. Litmas milk Good, perlcle None Ooa gulzggr g g l gig fiig POSI- Ve Gelatin stab Poor Grayish white Poor, grayish white do Liquefaction, posi iv This invention relates to a novel method for producing PHYSIOLOGICAL CHARACTERISTICS an antibiotic triostin ossessin otent antibacterial ro erties p g P p p (1) Optlmum temperature: 28 C.

I o o In particular, the invention relates to a method for (2) g i growth at 45 producing an antibiotic, triostin, characterized by utilizai i i g6 19 95. we tion of a new microorganism, Streptomyces triostinicus, y ysls 0 i PO51 n. (5) Tyrosinase reaction: negative The antibiotic, triostin, was previously isolated by the (6) peptomzanon of P present inventor from a cultured broth of Streptomyces (7) coagulfltlon posltlfe S-2-2l0, and it was also reported that the triostin com- (8) F P P of mate: negat1ve plex consists substantially of three components, i.e. trio- (9) Utlhzatlon Fenulose: negatlve stins A, B and C and that triostin C has potent anti- (10) chromoaemcltw negallve bacterial properties, especially against gram-positive bac- (11) Productlon of melanold P negative teria Journal of Antibiotics, S61. A, (1961) Now, it has newly been discovered that a microorganism belonging to a new species, Streptomyces trio- Carbon source: Result stinicus, is capable of producing the said antibiotics when Glucose cultivated in an aqueous nutrient medium under a sub- MannoSe merged aerobic condition in a higher yield than that D fructose achieved with the known microorganism, Streptomyces S- Inositol 2-210. The present invention has been accomplished on Larabinose the basis of this discovery. Lactose Accordingly, the principal object of the invention is 65 Rh to provide a new method for the production of the anti- Rafiinose biotics, triostins. Another object of the invention is to Salicin provide a new microorganism being capable of producing D-rnannrtol useful antibiotics in an excellent yield. A further object L-XylOSe i of the invention is to provide an effective improvement Sucrose 3v Note: good growth and positive utilization; moderate growth and positive utilization; i faint growth and probably no-utilization; no growth and no-utilization.

Among the many species of Streptomyces described the microorganism in chromogenicity and productions of melanoid pigment and soluble pigment. However, the strain can be differentiated from these known species by characteristics listed in the following table.

The pH tends to remain rather constant during the fermentation but, if variations are encountered, a buffering agent such as calcium carbonate may be added to the medium. Occasionally, the pH moves to the alkaline side during the fermentation and, in such case, an acidic substance such as acetic acid and ammonium sulfate may be added. Generally speaking, the pH may be kept between 5.9 and 8.2, preferably around 7. If excessive foaming is encountered during the fermentation anti-foaming aggpts such as vegetable oils, lard oil and polypropyleneglyc'ol may be added to the fermentation medium prior to or in the course of the fermentation. The maximum yields" of the antibiotic triostin can be obtained within about 72 to about 170 hours of fermentation under optimum conditions of temperature and aeration.

After growth of the microorganism, the mycelium may be removed from the fermentation broth by using standard equipment, such as filter-presses and centrifuges, and than the antibiotic triostin may be recovered from the COMPARISON WITH EVERY KNOWN SPECIES Streptomyces triostz'nicus Sireptomyces Streptomyces St. Strep Stu, Sh ATCC 21043 cacaoi acidophilus gougeroii pseudoar' griseo,

Aerial mycelium- Straight (a few Spirals Spirals Straight (short)- Spirals Straight.

loops or spirals).

Sucrosenitrate N o-growth. No-growth G, good; A, G, flat; A,

white to gray, white to gray; S, colorless. S, colorless.

Glucoseasparagin-.- G, good; A, gray- G, poor; A, yel- G, colorless; A, G, colorless; A, G, gray to pinkish white; S, low; S, none. gray, S, colorwhite; S, ish; A, none; none. less. colorless. colorless.

Nutrient agar G, brownish gray; G, brown; A, N o-growth G, green; A, G, colorless; A,

A, none; S, ivory; S, none. white; S, white; S, none. none. yellow.

Gelatin G, poor; A, none; A, none; S, none; S, colorless; L, S, cream; L, G, yellow A, none; L,

S, none; L, L, positive. positive. positive. brown; A, positive. positive. white to grey;

L, positive.

Milk G, colorless; P, No-growth G, white; L, G, orange; P, No-growth C, green; P,

positive; 0, positive; 0, positive; 0, positive; 0, positive. positive. positive. positive.

Potato G, brownish gray; G, brownish gray; G, colorless; G, colorless; A, G, colorless; A, G, light brown; G, glraysih; A,

A, none; 0, A, gray. PC, colorless. white; PC, white; PO, brown; A, w ite; PC, colorless. colorless. colorless. .white to gray; brown.

PC, brown.

Nora.G=growth; A=aerial mycelium; S=soluble pigment; L=liqueiaction of gelatin; P=peptionization of milk; C=coagulation of milk; PC=

plaque color.

As a result of the above observations, the microorganism of the present invention has been designated a new species and named Strepto'myces triostinicus, n. sp.

It is to be understood that for the production of triostin the present invention is not limited to the use of Streptomyces triostinicus, n. sp. It is especially desired and intended to include the use of triostin-producing mutants or variants produced from the described organism by ganism, Streptomyces triostinicus, n. sp., in an aqueous nutrient medium at a temperature of about 25 to about 37 C., preferably 27 to 29 C., under aerobic conditions. The composition of this nutrient medium maybe varied over a very wide range. Essentially what-is required is a carbon source, a nitrogen Source and trace inorganic elements. Examples of suitable carbon sources are starch, lactose, maltose, frustose, inositol and glycerol. Suitable sources of nitrogen for the fermentation process include meat extracts, peptone, corn steep liquor, soybean meal and yeast extracts. Examples of suit-able sources of inorganic elements are mineral salts, such as sodium chloride, potassium chloride, calcium carbonate and phosphates.

The nutrient medium may or may not be adjusted to about pH 7 prior to inoculation of the microorganism.

filtrate by a solvent extraction procedure. As the antibiotic triostin is retained by the mycelium in appreciable quantities, a solvent extraction procedure is preferably used to recover the antibiotic from the mycelium or the whole broth without the removal of the mycelium. Suitable extraction solvents include methanol, ethanol, dioxane, acetone, chloroform, tetrachloroethane and ethyl acetate. For the extraction of the antibiotic from larger volume of broth, however, an adsorption procedure is superior to an ordinary solvent extraction procedure. For instance, the whole broth may be filtered after the addition of an adsorbent, such as Hyflo Super-Ce] (diatomaceous earth), and the resulting cake of adsorbent and mycelium may be eluted with a suitable organic solvent, such as chloroform, dioxane, ethanol and methanol, to extract the antibiotic. The extract may be concentrated and a suitable organic solvent such as petroleum ether, petroleum benzin and ligroin added to precipitate the crude active component, i.e. triostin complex.

The thus obtained triostin complex may be further purified, if desired, by suitable operations, such as recrystallization, chromatography and the like, whereby the complex can be divided into its essential components, triostin A, triostin B and triostin C. Examples of suitable recrystallization solvent are methanol, ethanol,'acetone, ethyl acetate, chloroform and mixture thereof. The preferred chromatographic adsorbents are silica-gel, silicic acid and the like.

Triostins A, B and C are known substances, and their chemical structures have also been proposed (The Journal of Antibiotics, Ser. A, XIX, 128 (1966)). The main components are triostin A and triostin C, of which physi cal and chemical properties are as follows:

Triostin A: colorless needles, when recrystallized from chloroform-methanol; M.P. 245248 C. (decomp.); [a] 157i2 (c.=0.970 in chloroform);

3320, 2960, 1746, 1672, 1644, 1573, 1505, 1493, 1408, 1368, 1325, 1290, 1273, 1167, 1128, 1090; 1082, 1065, 1053, 1000, 980, 962, 939, 911, 834 cmr UVA Egg? 243 m (log 6: 4.85)

Analysis.Calcd. for C H O N S (percent): C, 55.21; H, 5.71; N, 15.47; S, 5.89. M.W., 1086. Found (percent): C, 55.25; H, 5.71; N, 15.47; S, 5.89. N.W., 1035 (osmometry, chloroform).

Triostin C: colorless needles, when recrystallized from methanol, ethyl acetate, chloroform and mixture thereof, and colorless plates, when recrystallized from acetonecontaining solvent; M.P. 210-214" C.; [oc] 143.9:L-2 (c.=1.121 in chloroform);

IRv Q3? 3420, 1745, 1690, 1650, 1578, 1515, 1495, 1410, 1245, 1205, 118 0, 1131, 110 2, 1059, 1038, 1014, 982, 932, 910, 829, 7-76, 775 cm.

UV). 352* 243 m;i(log e: 4.87), 315-326 m (log 6: 4.13).

Analysis.-Calcd. for C54H70012N12S2 (percent): C, 56.74; H, 6.13; N, 14.71; S, 5.60. Found (percent): C, 56.64; H, 6.23; N, 14.32; S, 5.21.

Triostin C shows inhibitory activity against a variety of microorganisms, especially against gram-positive bacteria in wide range and Mycobacterium tuberculosis var. hominis H37Rv, and the following table illustrates the anti-bacterial spectrum of triostin C.

MINIMUM INHIBITORY CONCENTRATION (MIC) MIG Test organisms (meg/ml.) Medium Staphylococcus aureus:

209 P 0.10 Nutrient agar. 'Ier shim 0.10 Do. Bacillus substilzs PCI 2 0.05 Do. Bacillus anthracis 0.1 Do. Bacillus agri 0. 05 Do. Sarcina lutea 0. 05 D: Diplococcus pueumouiae:

0. 02 Blood agar. 0.02 Do. Type II 0.02 Do. C'oryuebacterium diphtheriae:

Tr 0. 01 Do. 0.01 Do. Streptococcus hemolyticus:

0. 1 Do. HA 0.1 Do. Shigella dyseuteriae... 100. 0 Nutrient agar. Shigella sonnet 100. 0 Do. Salmonella typhosm. 100. 0 Do. Salmonella paratyphi 100. 0 Do. Escherichia coli 100. 0 D0. Pseudo'monas aerugiuosa 100. 0 D0. Klebsiella pneumoniae 100. 0 Do.

Mycobacterium tuberculosis var. homim's H87Rv 0.2 Kirchners medium.

The antibacterial spectrum of triostin complex is quite similar to that of triostin C. The median lethal dose value (LD value) was determined in mice intraperitoneally to be 100 mg./kg.

The antibiotic triostin complex and its individual components are useful as agents for inhibiting the growth of gram-positive pathogenic microorganisms and mycobacteria. It is useful for sterilizing equipment, for example surgical instruments. It is also useful in obtaining pure cultures of single organisms where a susceptible organism may be separated from a resistant one.

The following example is given solely for the purpose of illustration and is not to be construed as limitations of this invention, many variations of which are possible.

EXAMPLE Streptomyces triostinicus ATCC No. 21,043 is inoculated to a nutrient medium composed of 5 g. of meat extract, 5 g. of peptone, 20 g. of glucose, 0.5 g. of sodium chloride, 0.35 g. of calcium carbonate and 1 l. of distilled water, and incubated at 28 C. for 2 days. The thus obtained cultured broth is used as inoculum.

A nutrient medium composed of 20 g. of sodium nitrate, 10 g. of potassium hydrogen phosphate, 2.5 g. of calcium chloride, 20 g. of D-maltose and 10 l. of distilled water is sterilized and inoculated with the inoculum prepared above. The cultivation is performed at 28 C. for 3 to 7 days under an aerobic condition, until the concentration of triostin amounts to about ,ag/ml. (determined by spectrophotometer).

The cultured broth is filtered and the filtrate is extracted with 5 l. of ethyl acetate. On the other hand, the collected mycelium is 2 to 3 times extracted with a total of 4 l. of 70% aqueous acetone and the aqueous acetone solution is evaporated under reduced pressure. The aqueous residue is adjusted to an approximately neutral pH and extracted 2 to 3 times with a total of 4 l. of ethyl acetate. The ethyl acetate extracts are combined, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue is dissolved in chloroform or ethyl acetate and crude triostin complex is precipitated by addition of petroleum ether. Yield, 1.3 g.

The thus obtained triostin complex is dissolved in chloroform and chromatographed on 350 g. of silicic acid and eluted with chloroform-methanol (:1) to give 220 mg. of triostin C, a trace of triostin B and 400 mg. of triostin A.

What is claimed is: 1. A process for producing the antibiotic triostin which comprises cultivating a strain of Streptomyces triostinicus n. sp., ATCC No. 21,043, in an aqueous nutrient medium at a pH from about 5.9 to 8.2 and at a temperature from about 25 C. to 37 C. under aerobic conditions for about 3 to 7 days, and recovering the accumulated antibiotic from the culture medium.

2. A process according to claim 1 wherein the pH is about 7.

3. A process according to claim 1, wherein the temperature is about 27 C. to 29 C.

References Cited Chemical Abstracts, 'vol. 56, 1962, p. 7803g. Journal of Antibiotics, vol. 14, November 1961, pp.

JOSEPH M. GOLIAN, Primary Examiner 

